Synthesis of temozolomide and analogs

ABSTRACT

This invention relates to a novel process for the synthesis of Temozolomide, an antitumor compound, and analogs, and to intermediates useful in this novel process.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional application Ser.No. 60/262,465 filed Jan. 18, 2001.

FIELD OF THE INVENTION

This invention relates to a novel process for the synthesis ofTemozolomide, an antitumor compound, and analogs, and to intermediatesuseful in this novel process.

BACKGROUND OF THE INVENTION

Temozolomide, 3-methyl-8-aminocarbonyl-imidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one, is a known antitumor drug; see forexample Stevens et al., J. Med. Chem. 1984, 27, 196-201, and Wang etal., J. Chem. Soc., Chem. Commun., 1994, 1687-1688. It has the formula:

It is described in U.S. Pat. No. 5,260,291 (Lunt et al.) together withcompounds of broadly similar activity such as higher alkyl analogs atthe 3-position.

The synthesis of I by the process described in J. Med. Chem. 1984, 27,196-201 can be simply depicted as follows, even though the authorsmention that the cycloaddition of the methylisocyanate to the compoundof the formula (B) can proceed through two different intermediates:

In this process, 5-amino-1H-imidazole-4-carboxamide (A) is convertedinto 5-diazo-1H-imidazole-4-carboxamide (B), which is then cyclized withmethylisocyanate in dichloromethane to provide a high yield ofclinical-grade Temozolomide. However, this process requires isolation ofthe unstable and potentially dangerous5-diazo-1H-imidazole-4-carboxamide (B). Moreover, methylisocyanate is adifficult reagent to handle and ship, especially on the industrialscale, and indeed is better avoided in industrial manufacture.Furthermore, the cycloaddition of methylisocyanate requires a very longreaction time: Table I in J. Med. Chem. 1984, 27, 196-201, suggests 20days.

The production of I by the two processes described in J. Chem. Soc.,Chem. Commun., 1994, 1687-1688 provides a low overall yield from5-amino-1H-imidazole-4-carboxamide (A): less than 20% (unoptimized—about17% through 5-diazo-1H-imidazole-4-carboxamide (B) and about 15% through5-amino-N¹-(ethoxycarbonylmethyl)-1H-imidazole-1,4-dicarboxamide (C)):

Moreover, the unstable 5-diazo-1H-imidazole-4-carboxamide (B) still hasto be isolated in the branch of this process that uses it as anintermediate.

Clearly, therefore, there is a need for synthetic methods that are moreconvenient, especially on an industrial scale, and provide good yieldsof clinical-grade Temozolomide, or improve the preparation or use ofintermediates for the aforementioned processes.

SUMMARY OF THE INVENTION

The present invention provides, as one embodiment, a process for thepreparation of Temozolomide and lower alkyl analogs thereof having theformula:

wherein R is an alkyl group having from 1 to 6 carbon atoms, whichcomprises:

(a) diazotizing a compound of the formula:

wherein R is as defined above;

and Pg″ is a divalent protecting group that is readily removable byhydrolysis or hydrogenolysis; or two monovalent protecting groups Pgthat are readily removable by hydrolysis or hydrogenolysis; or a bulkymonovalent protecting group Pg that is readily removable by hydrolysisor hydrogenolysis, together with a hydrogen atom;

and thereafter (b) subjecting the resulting compound of the formula:

wherein Pg″ is as defined above, to hydrolysis or hydrogenolysis.

Step (a) is preferably carried out in an aqueous-organic solution with asource of nitrous acid, in particular in solution in an aqueous organicacid such as a lower alkanoic acid, especially acetic acid.Water-miscible solvents such as lower alkanols, THF and DMF can bepresent. The source of nitrous acid is preferably inorganic, e.g., analkali metal salt of nitrous acid, most preferably sodium nitrite. Thereaction is preferably carried out in the presence of a reagent thatpromotes the correct direction of cyclization, e.g., LiCl.

Step (b) is preferably carried out by hydrolysis with a strong mineralacid such as concentrated HCl or HBr, or HClO₄, CF₃SO₃H, or MeSO₃H, orespecially concentrated sulfuric acid, at a moderate temperature such as−20 to 50° C. In a particularly preferred embodiment, thereadily-removable protecting group is a 1,1-dimethylethyl group (at-butyl group), together with a hydrogen atom. Its bulk also helps topromote the correct direction of cyclization.

The invention also provides novel intermediates useful in thepreparation of Temozolomide, in particular the compounds of the formulae11, III, IV, V, and VI, and the salts thereof:

wherein Pg″ is a protecting group as defined above, especially suchcompounds wherein Pg″ is a 1,1-dimethylethyl group together with ahydrogen atom, Ar is an arylmethylene group, and R is a lower alkylgroup as hereinbefore defined, especially a methyl group. An especiallypreferred arylmethylene group is the diphenylmethylene group; preferredcompounds of the formulae II and III include the compounds of theformulae:

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It should be noted that the cyclization of the compound of the formulaII above, wherein Pg″ is a monovalent protecting group Pg together witha hydrogen atom, could in theory also proceed to the nitrogen atom ofthe carbamoyl group, and yield an undesired aza-hypoxanthine derivative.The presence of a bulky protecting group Pg promotes the desiredcyclization to the imidazo[5,1-d]-1,2,3,5-tetrazine nucleus ofTemozolomide. The presence of LiCl in the reaction medium also has abeneficial effect in promoting the desired cyclization. The completeblocking of the nitrogen atom by the use of a divalent protecting groupor two monovalent protecting groups also ensures that the cyclizationproceeds in the desired direction.

The 1,1-dimethylethyl group was formerly known as t-butyl, sometimesabbreviated to t-Bu, and this old form of the name is still used herein(for convenience and especially brevity) in some of the formulae hereinand in the semi-trivial names in the reaction schemes and in theExamples.

The alkyl group R is preferably an unbranched alkyl group, in particularone with 1 to 4 carbon atoms, preferably 1-butyl, 1-propyl, ethyl orespecially methyl. When R is methyl, the product of the formula I isTemozolomide itself.

A particularly preferred embodiment of the process according to theinvention is shown in the following scheme, and a more general versionof this scheme is described thereafter:

In the first step of this process,[(diphenylmethylene)amino]acetonitrile 3 is allowed to react with anisocyanate PgNCO where Pg is a monovalent protecting group as definedabove, to yield an acetamide 4. This reaction is conveniently effectedin the presence of a base and of an inert organic solvent, under aninert atmosphere, e.g., nitrogen, and at a ambient temperature orreduced temperature, e.g., ambient temperature to −100° C., preferablyambient temperature to −10° C. The base is preferably one having theformula PgOM, where M is an alkali metal; other bases that can be usedinclude tertiary amines such as triethylamine and ethyidiisopropylamine,alkali metal hydrides such as sodium and potassium hydride, and alkalimetal carbonates such as sodium and potassium carbonate. The organicsolvent is preferably methylene chloride; however, other solvents thatcan be used include ethers such as methyl-t-butylether, diethylether,THF and dioxane, methylcyanide, ethylacetate, and hydrocarbons such astoluene, hexane and heptane.

The protecting group Pg is preferably a bulky alkyl group, e.g., onethat is strongly branched at the carbon atom having the free valency,especially a 1,1-dimethylethyl group. Other possible monovalentprotecting groups, some of which can be removed by hydrolysis, whereasothers can be removed by hydrogenation, include benzyl (orphenylmethyl), especially two benzyl groups, trityl (ortriphenylmethyl), benzyloxycarbonyl, and 9-fluorenyl. Divalentprotecting groups that may be used include benzylidene (orphenylmethylene) and 9-fluorenylidene. Further examples of suitableamino-protecting groups, and their use and removal, are given in“Protective Groups in Organic Synthesis”, Theodora Greene and PeterWuts, John Wiley & Sons, New York, N.Y., second edition (1991).

A divalent protecting group Pg″ or two monovalent protecting groups Pg₂can be introduced by an analogous reaction in which the t-BuNCO isreplaced by a compound of the formula Pg″:N.CO.Cl, wherein Pg″ is adivalent protecting group or two monovalent protecting groups Pg; thisreaction is also effected in the presence of a base and an inert organicsolvent substantially as described above. The compound of the formulaPg″:N.CO.Cl can be prepared by reaction of an imine or amine of theformula Pg″:NH with phosgene.

In the second step of this process, the acetamide 4 is subjected tohydrolysis to remove the diphenylmethylene group (an example of thegroup Ar) on the imino nitrogen, preferably with mild acid in an aqueousor aqueous-organic system, especially a mild inorganic acid (such asdilute mineral acid, e.g. 1 N hydrochloric acid, hydrobromic acid orsulfuric acid) in an inert organic solvent such as ethyl acetate; theproduct is the acetamide 5, as an acid addition salt such as thehydrochloride, hydrobromide or sulfate. The hydrolysis is convenientlyeffected at 0° C. to moderately elevated temperature, e.g., 100° C.,especially ambient temperature up to 70° C.

In the third step of this process, a salt, e.g., the hydrochloride, ofthe acetamide 5 is condensed with a urea derivative 7 or with analogsthereof on which each methyl group has been replaced with a group R,wherein the two groups R are identical and each group R is as definedabove. The urea derivative can be replaced with precursors thereof,e.g., the N-R-urea wherein R is as defined above (especiallyN-methylurea), together with an orthoformate, e.g. ethyl orthoformate,to provide the imidazole 6. This reaction can be carried out at aboutambient temperature in the presence of an inert organic solvent and amild acidic catalyst. The catalyst can be an organic acid, preferably aweak acid such as a carboxylic acid, especially a lower alkanoic acidsuch as acetic acid; the solvent is for example t-BuOMe or preferablymethylene chloride. The organic solvent is preferably methylenechloride; however, other solvents that can be used include ethers suchas methyl-t-butylether, diethylether, THF and dioxane, methylcyanide,ethylacetate, DMF, DMSO, and hydrocarbons such as toluene, hexane andheptane. The reaction is preferably carried out at about ambienttemperature or somewhat lower or higher, e.g., −25 to 50° C., preferably0 to 35° C.

Urea derivatives necessary for the fourth step can be prepared bycondensation of the N-R-urea wherein R is as defined above, especiallyN-methylurea with an orthoester, especially an orthoformate; thusmethyl[[[(methylamino)carbonyl]amino]methylene]urea 7 can be prepared bycondensation of N-methylurea with ethyl orthoformate at elevatedtemperature and under an inert atmosphere; see Whitehead, C. W.; J. Am.Chem. Soc., 1953, 75, 671.

In the fourth step of this process, the imidazo[5,1-d]-1,2,3,5-tetrazinenucleus of Temozolomide is assembled by diazotization of the imidazole 6or N-R analog thereof, wherein R is as defined above; preferredconditions have been described above.

The reaction can also be effected in an organic solvent with an organicsource of nitrous acid, e.g., t-butyl or isopentyl nitrite with acarboxylic acid such as a lower alkanoic acid, e.g., acetic acid, and inan organic solvent such as a lower alkanol, DMF, THF, ethyl acetate, ora hydrocarbon such as toluene, hexane or heptane.

The reaction presumably proceeds through a diazonium salt, whichspontaneously cyclizes to the compound of the formula III.

In the fifth step of this process, Temozolomide or N-alkyl analogthereof (wherein the alkyl group has 1 to 6 carbon atoms) is produced byhydrolysis of the protected-Temozolomide 8 or protected-N-alkyl analogthereof; again, the conditions have been described above.

The protected-Temozolomide 8 or protected-N-alkyl analog thereof(wherein the alkyl group has 1 to 6 carbon atoms) is an example of acompound of the formula III. In general, hydrolysis to remove aprotecting group is preferably carried out under an inert atmosphere andat a moderate temperature, e.g., at about 0° C. to 50° C., preferablyabout ambient temperature, in an aqueous acid. Alternatively, thehydrolysis can be carried out in an inert organic solvent in which thereagents (the acid and the compound 8) are at least partly soluble, forexample, methylene chloride. Hydrogenolysis to remove a protecting groupis preferably carried out under an inert atmosphere and at a moderatetemperature, e.g., at about ambient temperature to about 60° C., in aninert organic solvent with hydrogen and a hydrogenation catalyst such asPd/C or Raney Ni.

The compound of the formula 3 is known and can be prepared by thefollowing known process:

(See, for example, O'Donnell, M. J.; Polt, R. L; J. Org. Chem., 1982,47, 2663; and O'Donnell, M. J.; Eckrich, T. M.; Tetrahedron Lett. 1978,47, 4625.) Aminoacetonitrile 1 (preferably as an acid addition salt,e.g., the hydrochloride) is condensed with imine 2 in the presence of ananhydrous, inert organic solvent and under an inert atmosphere. Theimine provides a protecting group for the amino group of theaminoacetonitrile, a group that is stable to alkali but can be readilyremoved with mild acid when no longer needed. An aralkylidene-imine,especially a diphenylmethylidene-imine, is convenient. The organicsolvent is conveniently methylene chloride.

In the compounds of the formula 3 and 4, the amino-protecting group(Ph)₂C: can be replaced with another appropriate protecting group of theformula Ar, where Ar is as hereinbefore defined. In the compounds of theformulae 4, 5, 6, and 8, the 1,1-dimethylethylamino group can bereplaced with another appropriate protected amino group Pg₂N—or Pg″:N—,where Pg and Pg″ are as hereinbefore defined.

The compound of the formula 5 can be prepared also by the followingnovel method:

2-Cyano-N-(1,1-dimethylethyl)acetamide 11 (Bhawal, B. M.; Khanapure, S.P.; Biehl, E. R.; Syn. Commun., 1990, 20, 3235) is allowed to react withnitrosyl chloride in an inert organic solvent such as CH₂Cl₂ or CHCl₃ atmoderate temperature (e.g., ambient temperature to −25° C., preferablyabout 0° C.). The resulting2-cyano-N-(1,1-dimethylethyl)-2-(hydroxyimino)acetamide 13 is isolatedand reduced, for example with sodium dithionite in an aqueous organicsolvent, but preferably with aluminum amalgam in water at moderatetemperature (e.g., ambient temperature to about 0° C., preferably about0° C.).

The compound of the formula 13 is a novel intermediate and is a featureof the invention. The compound of the formula 13 forms salts with strongbases, e.g., with alkali metals such as sodium, and these salts are alsoa feature of the invention. Further features of the invention includecompounds analogous to 13 wherein the 1,1-dimethylethylamino group isreplaced by a protected amino group Pg″N, where Pg″ is as hereinbeforedefined. Such compounds can be prepared analogously from the compound ofthe formula 10 and a compound of the formula HN:Pg″, wherein Pg″ is asdefined above, especially a divalent group such as benzylidene or9-fluorenylidene, or two monovalent groups Pg such as two benzyl groups,or a monovalent group such as benzyl, trityl, benzyloxycarbonyl, or9-fluorenyl, together with a hydrogen atom.

5-Amino-1H-imidazole4-carboxamide, the intermediate of the formula (A)described in the ‘Background of the Invention’, can be advantageouslyprepared (e.g., as its hydrochloride 16.HCl) by the two routes shown inScheme VI, wherein 17 is a novel intermediate, 14 is commerciallyavailable and a method for its preparation is given in U.S. Pat.5,003,099, and the preparation of 6 has been described above. Thesepresent an improvement also in the preparation of Temozolomide, sincethe starting material of the formula (A) (for the diazotization in bothSchemes I and II above) is made more readily and/or more cheaplyavailable. Again, if desired, the N-methyl groups in the compounds ofthe formulae 6, 7 and 15 can be replaced with larger groups R, wherein Ris as defined above.

In route (a), purified aminocyanoacetamide 14 (obtained for example byrecrystallization, e.g., from acetone) is condensed with a ureaderivative 7 or with analogs thereof in which each methyl group has beenreplaced with a group R, wherein the two groups R are identical and eachgroup R is as defined above. The urea derivative can be replaced withprecursors thereof, e.g., the N-R-urea wherein R is as defined above(especially N-methylurea), together with an orthoformate, e.g. ethylorthoformate, to provide the imidazole 15. This reaction can be carriedout as described above for the reaction of 5 with 7 or with precursorsof 7. Imidazole 15 (or an analog thereof in which the methyl group hasbeen replaced with a group R, wherein R is as defined above), can thenbe hydrolyzed with mild base, e.g., a tertiary organic base such astriethylamine or ethyldiisopropylamine in an inert organic solvent suchas a lower alkanol, e.g., methanol, and the product 16 can then beconverted into its acid addition salt by reaction with the appropriateacid, e.g., the hydrochloride of 16 (or other salt as described in thenext paragraph) by reaction with hydrochloric acid, preferably in aninert organic solvent such as a lower alkanol, e.g., methanol orethanol, an ether such as methyl-t-butylether, diethylether, THF ordioxane, methylcyanide, ethylacetate, or a hydrocarbon such as toluene,hexane or heptane.

In route (b), imidazole 6 (or an analog thereof in which the methylgroup has been replaced with a group R, wherein R is as defined above)can be converted into another imidazole derivative 17 by hydrolysis witha mild base as described above for the first step of the conversion ofimidazole 15 into 16.HCl; and the free base can then be subjected toremoval of the protecting 1,1-dimethylethylamino group, and converted inthe same step into an acid addition salt, e.g., 16.HCl, preferably underconditions as described above for the second step of the conversion ofimidazole 15 into 16.HCl. The acid used in this step is preferably astrong acid, e.g., a mineral acid such as HCl (to provide 16.HCl), orHBr, H₂SO₄, HClO₄ or HNO₃, or a strong organic acid such as CF₃SO₃H orCH₃SO₃H. The solvent may be aqueous or, especially when the acid isCF₃SO₃H or CH₃SO₃H, organic.

The compound of the formula 6 is named as a starting material in thenovel process according to the invention, but can also be used as anovel starting material for the intermediates in the known process forthe preparation of Temozolomide. Further compounds that can be used inboth these aspects include compounds analogous to the compound of theformula 6 and having the formula II, and higher alkyl analogs of thecompound of the formula 6.

Preferred intermediates of the formulae VI and V include:

wherein Ar is as hereinbefore defined but is preferably adiphenylmethylene group.

In the compounds of the formulae 4, 5, 6, 8, 11, 13 and 17, and also thecompound of the formula t-Bu.NH.CO.C(N:Ar).CN, the1,1-dimethylethylamino group can be replaced with a protected aminogroup PgNH, Pg₂N, or Pg″N, where Pg and Pg″ are as hereinbefore defined.

The invention also provides a process for the preparation of a compoundof the formula IV, which comprises:

1. amidation of the ester group with a protecting amine, preferably1,1-dimethylethylamine and especially in the presence of a basiccatalyst and an inert organic solvent;

2. nitrosylation of the reactive methylene group, e.g., with an alkalimetal nitrite, e.g., sodium nitrite, and a weak acid such as an organicacid, especially acetic acid, but preferably with nitrosyl chloride inan inert organic solvent such as methylene chloride.

The resulting compound has the formula Pg″N.CO.C(:NOH).CN wherein Pg″ isa protecting group, especially a 1,1-dimethylethyl group together with ahydrogen atom. Compounds of this formula and the intermediates of theformulae Pg″N.CO.CH₂.CN and Pg″N.CO.CH(N:Ar).CN are also features of theinvention, especially those wherein Pg″ is a 1,1-dimethylethyl group,together with a hydrogen atom.

The invention also provides a process for the preparation of theabove-mentioned compound of the formula 8, which comprises diazotizing acompound of the formula II wherein Pg″N is a 1,1-dimethylethylaminogroup together with a hydrogen atom. This reaction can be effected underthe reaction conditions set out under paragraph (a) at the start of thesection Summary of the Invention.

Compounds of the formulae II, III, V and VI can exist in the form oftheir salts, for example with mineral acids, especially withhydrochloric acid and sulfuric acid. A particularly preferred salt ofthis type is compound 5.HCl.

Compounds of the formulae IV can exist in the form of their salts withbases, for example with alkali metals such as sodium.

The invention is not restricted to the specific embodiments of theprocesses shown in the foregoing Schemes III to VI and the specificintermediates used therein, but further comprises analogous processeswhich are carried out under different but substantially equivalentconditions, and also analogous processes and intermediates whereindifferent but broadly equivalent protecting groups Pg″ and Ar are used,and especially those wherein the methyl group (the precursor of the3-methyl group in Temozolomide) is replaced with a larger alkyl group R,wherein R is as hereinabove defined. Furthermore, the intermediates 4,5, 6, 8, 13, and 17, which are novel, can also be modified to includedifferent but broadly equivalent protecting groups Pg or Pg″ and Ar, andintermediates 6 and 8 can be modified to include a larger alkyl group R(wherein Pg, Pg″, Ar and R are as hereinabove defined). All theseembodiments are features of the present invention.

It should be noted that the unfused imidazole nucleus can generallyexist in two tautomeric forms (whose interconversion is catalyzed byacids), as illustrated in the following scheme for the compound offormula (A) above:

Although one such form may predominate, both formulae of such compoundsare generally covered in the description and claims of thisspecification, even where the name or formula specifically identifiesonly one.

When used herein, the following terms have the indicated meanings:

alkyl—represents a saturated hydrocarbon group having 1 to 6 carbonatoms, preferably 1 to 4, which may be straight or branched but ispreferably unbranched, e.g., 1-butyl, 1-propyl, ethyl, or especiallymethyl;

arylmethylene—represents a methylene group in which at least one arylgroup as defined below is substituted for at least one of the methylenehydrogen atoms. In compounds such as that of formula V, the methylenecarbon atom of the arylmethylene group is doubly bonded to the adjacentnitrogen atom. Representative arylmethylene groups includediphenylmethylene, phenylmethylene, and 9-fluorenylidene;

aryl (including the aryl portion of arylmethylene)—represents acarbocyclic group having from 6 to 14 carbon atoms and having at leastone fused benzenoid ring, with all available substitutable carbon atomsof the carbocyclic group being intended as possible points ofattachment, said carbocyclic group being optionally substituted with 1to 3 Y groups, where each group Y is independently selected from halo,alkyl, nitro, alkoxy and dialkylamino groups. Preferred aryl groups arephenyl, substituted phenyl, 1-naphthyl, 2-naphthyl and indanyl.

EXAMPLES

The following Examples illustrate but do not in any way limit thepresent invention:

Example 1: 3-Methyl-8-aminocarbonyl-imidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one (Temozolomide)

Step A: Preparation of2-cyano-N-(1,1-dimethylethyl)-2-[(diphenylmethylene)amino]-acetamide

The imine 3 (700 g, 3.178 mol) and CH₂Cl₂ (7 L) were placed into a 22 Lthree-necked flask equipped with a nitrogen inlet, a gas outlet tube,reflux condenser, thermometer, mechanical stirrer, and maintained undera positive pressure of nitrogen. 1,1-Dimethylethyl-isocyanate (442 mL,3.870 mol) was added to this stirred mixture at 0° C., and afterstirring for 10 min a solution of potassium t-butoxide in THF (1.0 M inTHF, 3.88 L, 3.88 mol) (as supplied by Aldrich) was added slowly (1hour). The solution was stirred at 0° C. for 4 hours, when the reactionmixture had become a very thick paste with a deep brown color, and thinlayer chromatography (EtOAc/hexanes=1/4) indicated that no more startingmaterial was present. The resulting mixture was quenched with saturatedNH₄Cl solution (5 L), and the organic layer was separated and washedsequentially with saturated NH₄Cl solution (5 L), and brine (5 L). Thecombined aqueous solution was extracted with CH₂Cl₂ (1 L). The combinedCH₂Cl₂ solutions were dried over MgSO₄ and concentrated under reducedpressure to yield a brown solid. The resulting crudeN-(1,1-dimethylethyl)-acetamide derivative was purified by slurrying inhexane (2.5 L) at a concentration of 1-5% at room temperature. Theslurry was filtered and the filter cake dried in a vacuum oven (20 mmHg, 20° C., 18 hours) to yield 0.914 kg (2.862 mol, 90%)N-(1,1-dimethylethyl)-acetamide derivative 4 as a brownish solid.

¹H NMR (400 MHz, CDCl₃, δ): 7.62 (d, 2 H), 7.53 (m, 4 H), 7.41 (m, 2 H),7.22 (m, 2 H), 4.62 (s, 1 H), 1.41 (s, 9 H); mp: 107-108° C.Step B: Preparation of 2-amino-2-cyano-N-(1,1-dimethylethyl)-acetamidehydrochloride, 5.HCl

2-Cyano-N-(1,1-dimethylethyl)-2-[(diphenylmethylene)amino]acetamide 4(900 g. 2.818 mol), ethyl acetate (4.5 L) and aqueous HCl (1 N. 4.5 L)were placed into a 12 L three-necked flask equipped with a nitrogeninlet, a gas outlet tube, reflux condenser, thermometer, mechanicalstirrer, and maintained under a positive pressure of nitrogen. Themixture was heated on an oil bath at 60° C. for 4 hours with vigorousstirring, gradually cooled to room temperature, and then slowly dilutedwith CH₂Cl₂ (4 L). (Thin layer chromatography (EtOAc/hexanes=1/4)indicated no more starting material was present.) The resulting layerswere separated and the aqueous phase was extracted with CH₂Cl₂ (4 L).The combined organic solutions were extracted with aqueous HCl (1 N,2×0.5 L). The aqueous extracts were combined and concentrated underreduced pressure to yield 490 g (2.557 mol) of2-amino-2-cyano-N-(1,1-dimethylethyl)-acetamide hydrochloride 5.HCl.

¹H NMR (400 MHz, DMSO, δ): 9.38 (bs, 2 H), 8.92 (s, 1 H), 5.28 (s, 1 H),1.30 (s, 9 H); mp: 211° C. (dec.)Step G: Preparation of5-Amino-N⁴-dimethylethyl)-N¹-methyl-1H-imidazole-1,4-dicarboxamide 6

2-Amino-2-cyano-N-(1,1-dimethylethyl)-acetamide hydrochloride 5.HCl (414g, 2.160 mol), urea 7 (414 g, 2.617 mol) (Whitehead, C. W.; J. Am. Chem.Soc., 1953, 75, 671), CH₂Cl₂ (4 L) and acetic acid (20 mL) were placedinto a 10 L, three-necked flask equipped with a nitrogen inlet, a gasoutlet tube, reflux condenser, thermometer, mechanical stirrer, andmaintained under a positive pressure of nitrogen. The mixture wasstirred vigorously at room temperature for 18 hours and thenconcentrated under reduced pressure. The residue was treated with H₂O (3L) and stirred for 30 min, and the solids were collected by vacuumfiltration. The solid was dried in an oven (20 mm Hg, 20° C., 18 hours)to yield 240 g of a grayish solid (0.943 mol, 94% pure, HPLC analysis).A standard sample of5-amino-N⁴-(1,1-dimethylethyl-N¹-methyl-1H-imidazole-1,4-dicarboxamide 6was prepared by recrystallization from EtOAc; mp: 145-147° C.

The aqueous solution was extracted with CH₂Cl₂ (2 L), the organicextract concentrated under reduced pressure, and the residue was washedsequentially with H₂O (200 mL) and EtOAc/hexanes (1/9, 500 mL) to yield130 g of additional grayish product (0.505 mol, 93% pure, HPLC assay).

¹H NMR (400 MHz, CDCl₃, δ): 7.45 (s, 1 H), 6.98 (bs, 1 H), 6.50 (s, 1H), 5.92 (bs, 2 H), 2.92 (d, 3 H), 1.40 (s, 9 H).

Although it was observed that smaller-scale reactions (using 1-15 g of5) gave higher percentage yields of relatively purer product (e.g.,90-95% yield, 93-98% pure), such small-scale reactions are lesspractical for the preparation of a commercial product.

Purification of5-Amino-N⁴-(1,1-dimethylethyl)-N¹-methyl-1H-imidazole-1,4-dicarboxamide6

5-Amino-N⁴-(1,1-dimethylethyl)-N¹-methyl-1H-imidazole-1,4-dicarboxamide6 (313 g, 93% pure by HPLC analysis) was suspended in EtOAc (4 L) andrefluxed for 10 min. The solution was filtered while hot to remove solidresidue, and was then cooled slowly to room temperature. The resultingsolid product was collected by vacuum filtration. The filtrate wasconcentrated under reduced pressure to a thick paste and then filteredto afford an additional solid product. The combined solids were purifiedby slurrying in t-BuOMe/2-PrOH (1.5 L, 9/1) at room temperature for 1hour. The solid product 6 was collected by filtration and was dried in avacuum oven (20 mm Hg, room temperature, 48 hours) to yield 252 g of atan-colored solid (98% pure against a standard sample by HPLC analysis).A satisfactory ¹H NMR spectrum was obtained.Step D:3,4-Dihydro-N-(1,1-dimethylethyl)-3-methyl-imidazo[5,1-d]-1,2,3,5-tetrazine-8-carboxamide8 (t-butyl-Temozolomide)

LiCl (45 g, 1.066 mol) (Aldrich), distilled H₂O (100 mL) and glacialacetic acid (2.5 mL, 43.9 mmol) were placed into a 500 ml three-neckedflask equipped with an overhead mechanical stirrer and thermometer. Thewarm solution was stirred for 30 min in an ice bath until cooled to roomtemperature.5-Amino-N⁴-(1,1-dimethylethyl)-N¹-methyl-1H-imidazole-1,4-dicarboxamide6 (5.0 g, 20.9 mmol, 98% pure) was then added, the mixture was stirredfor 30 min, and then NaNO₂ (1.9 g, 23 mmol) (Fischer) was added. Thereaction mixture was stirred at 0° C. for one hour and then at roomtemperature for 5 hours (when HPLC indicated that no more startingmaterial was present), and then diluted with CH₂Cl₂ (100 mL). Theresulting layers were separated and the aqueous phase was extracted withCH₂Cl₂ (100 mL). The combined organic solutions were washed with aqueousNa₂S₂O₄ (10 g/100 ml) and then with aqueous NaHCO₃ (saturated, 100 mL).The organic solution was concentrated under reduced pressure to afford 2as a yellow-brown solid (4.56 g, 88% pure, HPLC assay). A standardsample was prepared by flash chromatography (6:4, EtOAc:hexane) twice.Satisfactory ¹H and ¹³C NMR spectra and elemental analyses wereobtained; mp: 135-136° C.

¹H NMR (400 MHz, CDCl₃, δ): 8.38 (s, 1H), 7.20 (bs, 1 H), 4.04 (s, 3 H),1.52 (s, 9 H).Step E:3-Methyl-8-aminocarbonyl-imidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one 9(Temozolomide)

t-Butyl-Temozolomide 8 (4.01 g, 16.023 mmol) and conc. H₂SO₄ (8 mL)(Fisher Scientific) were placed into a 50 mL flask equipped with astirrer bar. The mixture was stirred for 2 hours at room temperature andthen slowly poured into ice-cold EtOH (160 mL). A white precipitateformed, which was collected by vacuum filtration and washed withice-cold EtOH (10 mL). The solid was dried under vacuum (20 mm Hg, roomtemperature, 72 hours) to yield 2.63 g of 9 (13.546 mmol, 98.4% pureagainst a standard sample by HPLC analysis)

The mother liquors contained an additional 9.7% of 9 (HPLC assay).

Example 2: Preparation of Intermediates and Reagents:

Part A: 2-Cyano-N-(1,1-dimethylethyl)-2-(hydroxyimino)acetamide 13

Amide 11 (3.11 g, 22.18 mmol) (Bhawal, B. M.; Khanapure, S. P.; Biehl,E. R.; Syn. Commun., 1990, 20, 3235) dissolved in CH₂Cl₂ (100 mL) wasplaced into a 500 mL 3-necked round-bottom flask equipped with astirring bar. The solution was cooled to 0° C. (ice bath) and NOCl(Fluka) was bubbled through until the reaction mixture turned abrick-red color. The reaction mixture was stirred at 0° C. for 30 minand then at room temperature for 18 hours. The precipitate was collectedand washed with CH₂Cl₂ (25 mL) to afford the product as a white solid(2.88 g, 17.0 mmol).

¹H NMR (400 MHz, DMSO-d₆, δ): 7.70 (s, 1 H), 3.32 (s, 1 H), 1.32 (s, 9H); mp: 218-219° C.Part B: 2-Amino-2-cyano-N-(1,1-dimethylethyl)acetamide 13

Oxime 13 (2.5 g, 14.78 mmol), Al amalgam (0.81 g) and distilled H₂O (100mL) were placed into a 250 mL round-bottom flask equipped with astirring bar, nitrogen inlet, a gas outlet tube, and maintained underpositive pressure of nitrogen. The reaction mixture was stirred at 2-10°C. (ice bath) for 2.5 hours, filtered, the filtrate extracted withCH₂Cl₂ (2×60 mL), and the combined organic layers were concentratedunder reduced pressure to afford the product as an oil (1.62 g, 10.44mmol). Concentration of the aqueous layer under reduced pressureafforded additional product 5 (0.41 g, 2.64 mmol).

¹H NMR (400 MHz, DMSO, δ): 7.68 (s, 1 H), 4.34 (s, 1 H), 2.78 (bs, 2 H),1.32 (s, 9 H).

The Al amalgam used in this Step was prepared as follows: HgCl₂ (1.6 g,5.89 mmol) was dissolved in 160 mL distilled H₂O in a 250 mLround-bottom flask equipped with a stirring bar. The solution was cooledto 0-5° C. (ice bath), aluminum foil (4.0 g, 148.3 mmol), cut into smallsquares (0.5 to 1.0 cm²), was added, and the mixture was stirred for 1.5min. It was then filtered, and the solids were washed with MeOH (2×60mL) and then t-BuOMe (60 mL), dried under vacuum (20 mm Hg, 3 hours) andstored under N₂.Part C: Purification of Aminocyanoacetamide 14

Aminocyanoacetamide 14 (60.0 g, 0.606 mol) (Aldrich, black solid) andacetone (2 L) were placed into a 5 L, three-necked flask equipped with anitrogen inlet, a gas outlet tube, reflux condenser, thermometer,mechanical stirrer, and maintained under a positive pressure ofnitrogen. The mixture was heated to reflux for 10 min with vigorousstirring, gradually cooled to room temperature, and then filtered. Theorganic solution was concentrated under reduced pressure to yield 55.2 g(0.557 mol) of 14. The product was dried in a vacuum oven (20 mm Hg, 20°C., 18 hours) and is a tan solid.Part D: Preparation of 5-Amino-N¹-methyl-1H-imidazole-1,4-dicarboxamide15

Aminocyanoacetamide 14 (80 g, 0.807 mol), urea 7 (139 g. 0.879 mol), andglacial acetic acid (0.96 L, 16.77 mol) (Fisher Scientific) were placedinto a 2 L, three-necked flask equipped with a nitrogen inlet, a gasoutlet tube, reflux condenser, thermometer, mechanical stirrer, andmaintained under a positive pressure of nitrogen. The mixture wasstirred vigorously at room temperature for 2 hours and then concentratedunder reduced pressure. After removal of most of the acetic acid, 200 mLof t-BuOMe was added and the mixture was concentrated under reducedpressure. The residue (a viscous oil) was treated with MeOH/t-BuOMe(1:20, 2.5 L), and precipitation was induced by scratching the glasssurface. The mixture was stirred for 30 min and the precipitate wascollected by vacuum filtration. The solid was dried in an oven (20 mmHg, 20° C., 18 hours) to yield 135 g of a grayish solid. The crudeproduct was purified by slurrying in H₂O (0.7 L) at room temperature for1 hour. The solid product 15 was collected by filtration and was ovendried (20 mm Hg, 20° C., 18 hours) to yield 129 g of a grayish solid(0.680 mol, 97% pure against a standard sample by HPLC analysis). Astandard sample of 5-amino-N¹-methyl-1H-imidazole-1,4-dicarboxamide 15was prepared by recrystallization from CH₃CN/H₂O (1:6); mp: 165-169° C.

¹H NMR (400 MHz, DMSO-d₆, δ): 8.50 (q, 1H), 7.67 (s, 1 H), 6.9 (bd, 2H), 2.83 (d, 3 H).

Part E: Preparation of 5-Amino-1H-imidazole-4-carboxamide hydrochloride16.HCl from 5-Amino-N¹-methyl-1H-imidazole-1,4-dicarboxamide 15

5-Amino-N¹-methyl-1H-imidazole-1,4-dicarboxamide 15 (10.72 g, 0.057 mol,97% pure against a standard sample by HPLC analysis), Et₃N (5 mL) andMeOH (100 mL) were placed into a 250 mL round-bottom flask equipped witha magnetic stir bar. The heterogeneous reaction mixture was heated at80° C. (oil bath) for 4 hour with vigorous stirring, gradually cooled toroom temperature (the reaction mixture is a dark homogeneous solution),and concentrated under reduced pressure. The residue (a viscous oil) wastreated with t-BuOMe/acetone/MeOH (50 mL/20 mL/5 mL) and stirred for 2hour. Precipitation was induced by scratching the glass surface. Theprecipitate was collected by vacuum filtration to yield 7.21 g of5-amino-1H-imidazole-4-carboxamide (as free base). The free base wasconverted into 5-amino-1H-imidazole-4-carboxamide hydrochloride 16.HClby slurrying in HCl/MeOH (2.6 M, 40 mL, 0.104 mol, prepared by bubblingHCl gas into MeOH). The solid product 16.HCl was collected by filtrationand air dried (2 hour) to yield 8.5 g of product (0.051 mmol, 97% pureagainst an Aldrich sample by HPLC analysis).

¹H NMR (400 MHz, D₂O, δ): 8.21 (s, 1 H).

Part F: Preparation of5-Amino-N-(1,1-dimethylethyl)-1H-imidazole-4-carboxamide 17

5-Amino-N⁴-(1,1-dimethylethyl)-N¹-methyl-1H-imidazole-1,4-dicarboxamide6 (10.4 g, 0.041 mol, 93% pure), MeOH (100 mL) and Et₃N (5 mL) wereplaced into a 250 mL, three-necked flask equipped with a nitrogen inlet,a gas outlet tube, reflux condenser, thermometer, magnetic stirrer bar,and maintained under a positive pressure of nitrogen. The mixture washeated at 80° C. (oil bath) for 3 hour with vigorous stirring (when HPLCanalysis indicated that no more starting material was present),gradually cooled to room temperature, and concentrated under reducedpressure. The gummy residue was treated with a solution of t-BuOMe (10mL), n-heptane (100 mL) and acetone (2 mL), and stirred at roomtemperature for 1 hour.

The resulting precipitate was collected by vacuum filtration and dried(20 mm Hg. 20° C., 18 hours) to yield 8.9 g (theoretical yield is 7.37g) of 5-amino-N-(1,1-dimethyl-ethyl)-1H-imidazole-4-carboxamide 17 as atan solid.

¹H NMR (400 MHz, CDCl₃, δ) 7.10 (s, 1 H), 6.80 (s, 1 H), 2.92 (d, 3 H).1.42 (s, 9 H); mp: 186° C. (dec.)

Part G: Preparation of 5-Amino-1H-imidazole-4-carboxamide hydrochloride16

5-Amino-N-(1,1-dimethylethyl)-1H-imidazole-4-carboxamide 17 (8.9 g,theoretical amount is 7.37 g, 0.041 mol) and conc. HCl (20 mL) wereplaced into a 100 mL, three-necked flask equipped with a nitrogen inlet,a gas outlet tube, reflux condenser, thermometer, magnetic stirrer bar,and maintained under a positive pressure of nitrogen. The mixture washeated at 80° C. (oil bath) for 1 hour with vigorous stirring, graduallycooled to 0° C., yielding a precipitate, and then slowly added to 2-PrOH(30 mL). The solids were collected by vacuum filtration and washed with2-PrOH (15 mL) to yield 4.97 g of product (0.030 mol, 97.5% pure againstan Aldrich sample by HPLC analysis). The filtrate was concentrated underreduced pressure to give a gummy residue. The gummy residue was treatedwith MeOH (20 mL) and stirred for 20 min. The solids were collected byvacuum filtration and washed with MeOH (10 mL) to yield an additional0.65 g of product (0.004 mol, 95% pure against an Aldrich sample by HPLCanalysis). The combined amount of 5-amino-1H-imidazole-4-carboxamidehydrochloride 16.HCl was 5.62 g (0.034 mol, 97% pure against an Aldrichsample by HPLC analysis).

¹H NMR (400 MHz, D₂O, δ): 8.21 (s, 1 H).

Analogs of Temozolomide, for example the 3-ethyl, 3-(1-propyl),3-(1-butyl), and 3-(1-hexyl) analogs, can be prepared by similarmethods.

All publications and patents cited herein are incorporated by referenceto the same extent as if each individual publication or patent wasspecifically and individually indicated to be incorporated by reference.

Whereas a number of embodiments of this invention are described herein,it is apparent that these embodiments can be altered to provide otherembodiments that utilize the compositions and processes of thisinvention. Therefore, it will be understood that the scope of thisinvention includes alternative embodiments and variations which aredefined in the foregoing specification and by the claims appendedhereto; and the invention is not to be limited to the specificembodiments presented herein by way of example.

1. A process for the preparation of a compound of the formula:

wherein R is an alkyl group having from 1 to 6 carbon atoms, whichcomprises: (a) diazotizing a compound of the formula:

wherein R is as defined above; and Pg″ is a divalent protecting groupthat is readily removable by hydrolysis or hydrogenolysis; or twomonovalent protecting groups Pg that are readily removable by hydrolysisor hydrogenolysis; or a bulky monovalent protecting group Pg that isreadily removable by hydrolysis or hydrogenolysis together with ahydrogen atom; and thereafter (b) hydrolyzing the resulting compound ofthe formula:


2. A process as claimed in claim 1 wherein R is a straight-chain alkylgroup having from 1 to 4 carbon atoms.
 3. A process as claimed in claim1 wherein R is a methyl group.
 4. A process as claimed in claim 3wherein Pg″ is a monovalent protecting group together with a hydrogenatom.
 5. A process as claimed in claim 4 wherein the monovalentprotecting group is a 1,1-dimethylethyl group.
 6. A process as claimedin claim 5 wherein step (a) is carried out in solution in an aqueousorganic acid with a source of nitrous acid.
 7. A process as claimed inclaim 6 wherein the organic acid is acetic acid and the source ofnitrous acid is inorganic.
 8. A process as claimed in claim 7 whereinthe source of nitrous acid is sodium nitrite.
 9. A process as claimed inclaim 8 wherein the reaction is carried out in the presence of LiCl. 10.A process as claimed in claim 5 wherein step (b) is carried out byhydrolysis with a mineral acid.
 11. A process as claimed in claim 10wherein the mineral acid is concentrated sulfuric acid.
 12. A process asclaimed in claim 1 for the preparation of Temozolomide having theformula:

which comprises (a) diazotizing a compound of the formula:

wherein Pg″ is a divalent protecting group that is readily removable byhydrolysis or hydrogenolysis; or two monovalent protecting groups Pgthat are readily removable by hydrolysis or hydrogenolysis; or a bulkymonovalent protecting group Pg that is readily removable by hydrolysisor hydrogenolysis, together with a hydrogen atom; and thereafter (b)subjecting the resulting compound of the formula:

wherein Pg″ is defined above, to hydrolysis or hydrogenolysis.
 13. Aprocess as claimed in claim 12 wherein the protecting group Pg″ is a1,1-dimethylethyl group together with a hydrogen atom, the diazotizationis effected in solution in acetic acid with sodium nitrite and in thepresence of LiCl;
 14. A process as claimed in claim 1 wherein thecompound of the formula II is prepared by reaction of a compound of theformula Pg″N.CO.CH(NH₂).CN (V) (wherein Pg″ is a protecting group asdefined in claim 1) withmethyl[[[(methylamino)carbonyl)amino]methylene]urea or with N-methylureaand an orthoformate in an inert organic solvent.
 15. A process asclaimed in claim 14 wherein the compound of the formula V is prepared byhydrolysis of a compound of the formula Pg″N.CO.CH(N:Ar).CN (VI)(wherein Pg is as defined in claim 14 and Ar is an arylmethylene group)with mild add.
 16. A process as claimed in claim 15 wherein Pg is a1,1-dimethyl group together with a hydrogen atom, and Ar is adiphenylmethylene group.
 17. A process as claimed in claim 15 whereinthe compound of the formula VI wherein Pg is a 1,1-dimethylethyl grouptogether with a hydrogen atom and Ar is a diphenylmethylene group isprepared by condensation of [(diphenylmethylene)amino]acetonitrile with1,1-dimethylethylisocyanate.
 18. A process for the preparation of acompound having the formula III set forth in claim 1, which comprisesdiazotizing a compound of the formula II set forth in claim
 1. 19. Aprocess for the preparation of a compound having the formula II setforth in claim 1, which comprises reacting a compound of the formulaPg″N.CO.CH(NH₂).CN (V) with a compound of the formula R.NH.CO.NH.CH:N.CO.NH.R or with an N-R-urea and an orthoformate in an inert organicsolvent (wherein Pg″ is a protecting group as defined in claim 1 and Ris as defined in claim 1).
 20. A process as claimed in claim 19, whichcomprises reacting a compound of the formula t-BuNH.CO.CH(NH₂).CN withmethyl[[[(methylamino)carbonyl]-amino]methylene]urea or withN-methylurea and an orthoformate in an inert organic solvent.
 21. Aprocess for the preparation of a compound having the formulaPg″N.CO.CH(NH₂).CN (V), which comprises hydrolyzing a compound of theformula Pg″N.CO.CH(N:Ar).CN (VI) (wherein Pg″ is a protecting group thatis readily removable by hydrolysis as defined in claim 1, and Ar is anarylmethylene group) with mild acid.
 22. A process for the preparationof a compound of formula I

where R is a methyl group I which comprises; (a) diazotizing a compoundof the formula:

in solution in acetic acid with an inorganic nitrate in presence ofLiCl, wherein; Pg″ is a monovalent protecting group together with ahydrogen atom wherein the monovalent protecting group is a1,1-dimethylethyl group: and thereafter (b) hydrolyzing the resultingcompound of the formula:


23. A process as claimed in claim 1 for the preparation of Temozolomidehaving the formula:

which comprises (a) diazotizing a compound of the formula in solution inacetic acid with sodium nitrite and in the presence of LiCl:

wherein Pg″ is a 1,1-dimethylethyl group together with a hydrogen atom,(b) subjecting the resulting compound of the formula:

wherein Pg″ is as defined above, to hydrolysis wherein said hydrolysisis carried out with concentrated sulfuric acid.